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Journal of Bioenergetics and Biomembranes

, Volume 12, Issue 3–4, pp 165–174 | Cite as

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

  • Joseph D. Robinson
Research Articles

Abstract

The (Na+ + K+)-dependent ATPase exhibits substrate sites with both high affinity (Km near 1 µM) and low affinity (Km near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl2, the nonhydrolyzable β-γ imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theKD for AMP-PNP was 4.2 µM in the absence of MgCl2. Adding 50 µM MgCl2, however, decreased theKD to 2.2 µM; by contrast, higher concentrations of MgCl2 increased theKD until, with 2 mM MgCl2, theKD was 6 µM. The half-maximal effect of MgCl2 on increasing theKD occurred at approximately 1 mM. This biphasic effect of MgCl2 is interpreted as Mg2+ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl2 concentrations would represent Mg2+ acting through the low-affinity substrate sites. NaCl in the absence of MgCl2 increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl2, however, NaCl increased theKD for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl2, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K+-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.

Keywords

Nucleotide MgCl2 Enzyme Preparation Simultaneous Presence Molar Excess 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Publishing Corporation 1980

Authors and Affiliations

  • Joseph D. Robinson
    • 1
  1. 1.Department of PharmacologyState University of New York Upstate Medical CenterSyracuse

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